Journal: EBioMedicine

Article Title: A truncating MEIOB mutation responsible for familial primary ovarian insufficiency abolishes its interaction with its partner SPATA22 and their recruitment to DNA double-strand breaks

doi: 10.1016/j.ebiom.2019.03.075

Figure Lengend Snippet: The MEIOB-truncating mutation disrupts MEIOB-SPATA22 recruitment to DNA damage foci. a-b- The truncated form of MEIOB is stable when expressed in HeLa cells. a- Western blot showing the expression of the wild-type (MEIOB MF ) and the truncated forms (MEIOB MF-mut ) of MEIOB transfected in HeLa cells. The ProSieve QuadColor protein marker 4,6 kDa–300 kDa (Lonza) was run on the left (Ladder). b- MEIOB MF or MEIOB MF-mut and SPATA22 were co-expressed from a single plasmid MEIOB∧SPATA22 in HeLa cells before exposure to etoposide (25 μM). Cells were fixed in PFA without pre-permeabilisation to retain both cytoplasmic and nuclear fractions. Antibodies were used to immunostain MEIOB (red) and SPATA22 (white). Yellow arrows highlight transfected cells expressing both MEIOB and SPATA22. c-d- The truncation prevents MEIOB recruitment with SPATA22 to DSBs. MEIOB MF or MEIOB MF-mut and SPATA22 HA were expressed in HeLa cells before exposure to etoposide. Cells were permeabilised before fixation in PFA to analyse the chromatin-associated fraction of proteins. RPA foci are formed at DNA damage sites. c- MEIOB or SPATA22 alone are not recruited to DNA damage sites. d- When co-expressed, MEIOB MF and SPATA22 are recruited to DNA-damage sites and colocalise with RPA. This colocalisation is not observed for the truncated MEIOB MF-mut . Antibodies are used to immunostain MEIOB (red), SPATA22 (white), and RPA (green). Scale bar = 10 μm in all panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: HeLa cells were used for minigene assays (CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030).

Techniques: Mutagenesis, Western Blot, Expressing, Transfection, Marker, Plasmid Preparation

Journal: Nature Communications

Article Title: Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin

doi: 10.1038/s41467-023-42714-4

Figure Lengend Snippet: a Activity of 3033 on JAK family enzymes. Human HEL 92.1.7 cells were treated with siRNA at 1.5 μM for 72 h, and mRNA levels of JAK1, JAK2, JAK3, and TYK2 were measured using the QuantiGene 2.0 assay ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, **** P < 0.0001, ns: not significant). b Ruxolitinib inhibits JAK1 and JAK2 to prevent the pathogenesis of vitiligo driven by IFN-γ signaling. c mRNA expression of IFN-γ-inducible chemokines CXCL9, 10, and 11. Cells were first treated with siRNA or ruxolitinib at 1.5 μM for 72 h, and then stimulated with recombinant human IFN-γ for 24 h ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). d , e Chemical engineering of 3033 for skin delivery. Terminal nucleotides were stabilized with phosphorothioate backbone modifications for nuclease stability ( d ). Human HeLa cells were transfected with DCA-siRNAs for 72 h through lipofectamine RNAiMax-mediated uptake and mRNA levels were measured using the QuantiGene 2.0 assay ( n = 3 biologically independent samples, mean ± s.d.; two-way ANOVA multiple comparison, * P < 0.05). f Molecular modeling (PyMOL 2) of DCA-siRNA 3033 in scaffold 2, si3033. g – i ruxolitinib and si3033 both reduce luciferase activity in IFN-γ-JAK1/2-STAT1 HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; one-way ANOVA, ** P < 0.01, **** P < 0.0001). Cells were treated with ruxolitinib or siRNA for 72 h and then stimulated with IFN-γ for 18 h. si3033 was transfected to the HeLa reporter cells using lipofectamine RNAiMax. j JAK1 and JAK2 mRNA expression in si3033-treated and untreated HeLa reporter cells ( n = 7 biologically independent samples, mean ± s.d.; two-sided unpaired t test, **** P < 0.0001, ns: not significant). Source data are provided as a Source Data file.

Article Snippet: GAS reporter (Luc)-HeLa cell line (IFN-γ/JAK/STAT1 pathway) was purchased from BPS Bioscience (#79041).

Techniques: Activity Assay, Expressing, Recombinant, Transfection, Comparison, Luciferase

Journal: Nature

Article Title: Experimental and computational framework for a dynamic protein atlas of human cell division

doi: 10.1038/s41586-018-0518-z

Figure Lengend Snippet: Quantitative imaging of mitotic proteins. (a) Automatic calibrated 3D live confocal imaging pipeline. Cells in prophase were identified by online classification, imaged through mitosis in the landmarks and protein of interest channels, and measured by FCS at selected positions. (b) The local protein concentrations determined by FCS fitting linearly correlate with the background subtracted image intensities at the corresponding positions (shown are data acquired on the same day). (c) Example cell showing concentration map resulting from FCS-based intensity calibration (mean z-projection). Scale bar: 10 μm. Data shown in (a)-(c) is for H2B-mCherry mNEDD1-LAP (EGFP) and is representative of n = 92 independent experiments performed with 28 different cell lines.

Article Snippet: HeLa Kyoto cell lines with H2B-mCherry were imaged live using an excitation laser at 561 nm every 5 min for about 16 hours on the Zeiss 780 microscopy system.

Techniques: Imaging, Concentration Assay

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Knockdown of FLNB promotes proliferation and inhibits apoptosis of HeLa cells. (A) Relative mRNA expression of FLNB in HeLa cells after it was knocked down using FLNB -specific shRNA was determined by reverse transcription-qPCR. shFLNB_1 and shFLNB_2 indicate two biological repeats of HeLa cells transfected with FLNB-specific shRNA. Ctrl_1 and Ctrl_2 indicate two biological repeats of HeLa cells transfected with empty vector as controls. (B) Cell proliferation of shFLNB was measured by an MTT assay in HeLa cells. Cell apoptosis of cells transfected with shFLNB was measured by (C) flow cytometry and (D) subsequent analysis by a 7-ADD and Annexin V assay. *P<0.05 and **P<0.01 vs. respective Ctrl. FLNB , filamin B; sh, short hairpin; qPCR, quantitative PCR; Ctrl, control; 7-AAD, 7-amino actinomycin D; OD, optical density; PE, phycoerythrin.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Knockdown, Expressing, shRNA, Reverse Transcription, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Annexin V Assay, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: GO and KEGG analysis of differentially expressed genes between short hairpin FLNB-transfected and control HeLa cells. Top 10 most enriched GO terms (biological process) of (A) upregulated and (B) downregulated genes upon FLNB knockdown. Rectangles around GO terms indicate notable cancer-related and cartilage development terms. Top 10 most enriched KEGG pathways of (C) upregulated and (D) downregulated genes upon FLNB knockdown. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FLNB , filamin B.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Transfection, Control, Knockdown

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Validation of FLNB -regulated genes (DEGs). (A) Relative expression level (FPKM, up) and RT-qPCR measurement (down) of cartilage development-related DEGs. (B) Related expression level (FPKM, up) and RT-qPCR measurement (down) of apoptotic-related DEGs. (C) Western blot analysis of two apoptotic-related proteins in shFLNB and Ctrl HeLa cells. FLNB , filamin B; DEGs, differentially expressed genes; FPKM, fragments per kilobase of transcript per million fragments mapped; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; Ctrl, control; ATP7A, ATPase copper transporting α; BMP7, bone morphogenetic protein 7; COL2A1, collagen type II α 1 chain; MMP13, matrix metallopeptidase 13; IL23A, interleukin 23 subunit α; MALAT1, metastasis associated lung adenocarcinoma transcript 1; NAIP, NLR family apoptosis inhibitory protein; SLC25A36, solute carrier family 25 member 36; MAP2K7, mitogen-activated protein kinase kinase 7.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Reports

Article Title: Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells

doi: 10.3892/or.2020.7532

Figure Lengend Snippet: Validation of FLNB -affected ASEs. Genome visualization (left panel) shows FLNB -regulated ASEs in shFLNB and control. (A) Validation of an ASE of VDR in HeLa cells. (B) Validation of an ASE of PACS2 in HeLa cells. (C) Validation of an ASE of MAP2K7 in HeLa cells. (D) Validation of an ASE of MALAT1 in HeLa cells. The number of junction reads were marked on the line representing splice junction composing ASE. The structures of ASEs are depicted in the top-right panel. The altered ratio of ASEs in RNA-sequencing and in reverse transcription-quantitative PCR were calculated and plotted (right panel, bottom). FLNB , filamin B; ASEs, alternative splicing events; sh/SH, short hairpin; NC, negative control; VDR, vitamin D receptor; PACS2, phosphofurin acidic cluster sorting protein 2; MAP2K7, mitogen-activated protein kinase kinase 7; MALAT1, metastasis associated lung adenocarcinoma transcript 1. *P<0.05 and ***P<0.001 vs. respectively NC.

Article Snippet: Human HeLa cells (CCTCC@GDC0009) were obtained from The China Center for Type Culture Collection.

Techniques: Biomarker Discovery, Control, RNA Sequencing, Reverse Transcription, Real-time Polymerase Chain Reaction, Alternative Splicing, Negative Control